Effects of Temperature on Growth Rate, Cell Composition and Nitrogen Metabolism in the Marine Diatom Thalassiosira Pseudonana (Bacillariophyceae)

MARINE ECOLOGY PROGRESS SERIES Vol. 225: 139–146, 2002 Published January 11 Mar Ecol Prog Ser

Effects of temperature on growth rate, cell composition and nitrogen metabolism in the marine diatom Thalassiosira pseudonana (Bacillariophyceae)

John A. Berges*, Diana E. Varela**, Paul J. Harrison

Department of Earth and Ocean Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

ABSTRACT: Although temperature effects on phytoplankton growth and photosynthesis can be clearly demonstrated in the laboratory, their relevance in the field is much harder to establish. Recently, however, it has been recognized that temperature has a significant influence on nitrogen uptake. In particular, temperate marine diatom species may be limited by their ability to acquire nitrate at temperatures above approximately 16°C. In order to explore this idea, we grew the diatom Thalassiosira pseudonana at 8, 17 and 25°C, and compared cell composition, and rates of growth (µ), 15N incorporation, calculated nitrate incorporation (the product of µ and cell N content), and the activity of nitrate reductase (NR), a key enzyme involved in nitrate incorporation. Cell N content, protein and volume remained relatively constant across different temperatures, but cell C, chlorophyll a (chl a), and C:N ratio increased with increasing temperature, suggesting that C metabolism was affected more strongly than N metabolism. Classical temperature models suggested that growth and various indices of nitrate metabolism all responded to temperature, with Q10 values of close to 2 over the whole temperature range. However, Q10 values over the interval from 8 to 17°C were higher than 2 and much lower than 2 between 17 and 25°C. Limitations to the Q10 concept are considered. Tem- perature effects on different measures of nitrate metabolism were very similar, supporting the hypothesis that the effects of temperature on diatom nitrate metabolism are mediated at the level of NR activity. Recent biochemical data for NR also supports this idea.

KEY WORDS: Nitrate reductase · Enzyme activity · 15N uptake · Phytoplankton ecology · Chemical composition · Temperature adaptation

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INTRODUCTION (1972) observation that ‘temperature does not seem to be very important in the production of phytoplankton in the Although there can be little doubt that temperature sea‘. Indeed, temperature does not even appear as a exerts a strong influence on algal growth and photosyn- variable in a recent review of phytoplankton primary thesis (e.g. Raven & Geider 1988, Davison 1991), biolog- productivity models (Behrenfeld & Falkowski 1997). ical oceanographers have tended to concur with Eppley’s Reasons for this apparent contradiction, include the fact that effects of temperature can be overshadowed by factors such as irradiance (e.g. Gibson & Foy 1989), and the **Present addresses: considerable ability of phytoplankton to acclimate or **School of Biology and Biochemistry, Queen’s University, adapt to growth at different temperatures (e.g. Li 1980, Belfast BT9 7BL, Northern Ireland, UK. **E-mail: [email protected] Smith et al. 1994, Suzuki & Takahashi 1995). **Marine Sciences Institute, University of California at Santa Temperature effects on algal metabolism extend Barbara, California 93106, USA beyond growth and photosynthesis, however. There is

a large body of literature showing that temperature level temperature is acting. Our hypothesis was that affects cell composition, short-term nutrient uptake observed changes in nitrate incorporation can be and, in particular, nitrogen metabolism (see Morris explained by changes at the biochemical level, i.e. in et al. 1974, Yoder 1979, Terry 1983, Raimbault 1984, NR activity. Whalen & Alexander 1984, Thompson et al. 1992, Reay et al. 1999, Sakamoto & Bryant 1999). These effects, while more subtle than those on growth and photosyn- MATERIALS AND METHODS thesis, can have enormous implications at the level of the ecosystem. Recently, for example, Lomas & Glibert Culture conditions. Cultures of the marine diatom (1999a,b) proposed that diatoms and diatom nitrate Thalassiosira pseudonana (Clone 3H) were obtained uptake are strongly limited by temperature; this has from the Northeast Pacific Culture Collection and important implications for species succession and the grown in 1 l semi-continuous batch cultures on artifi- biogeochemistry of nitrogen in marine environments. cial medium, under continuous light (150 µmol quanta In order to investigate such effects, it is necessary to m–2 s–1), as previously described (Berges & Harrison examine both cellular and subcellular processes. The 1993). All nutrients were in excess, and the sole nitro- effects of temperature are usually considered to have gen source (nitrate) was maintained at >20 µM at all their basis in altering enzyme-mediated biochemical times. Cultures were stirred and bubbled gently with processes. The relationship between temperature and air. Triplicate cultures were grown at either 8 ± 1°C, 17 a given biological rate can be modelled in several dif- ±1°C or 25 ± 1°C using a combination of a circulating ferent ways (see Ratkowsky et al. 1983, Ahlgren 1987), cooled water bath and immersion heaters. The semi- but the temperature coefficient Q10 (the factor by continuous cultures were acclimated for a minimum of which a biological rate is increased by a 10°C rise in 8 generations by diluting to one-sixth their original temperature) has been most commonly used. The use density as they neared the end of the logarithmic of Q10 values assumes an Arrhenius-type relationship phase of growth. Growth rates were monitored by in between rates and temperature and relies on chemical vivo fluorescence measured in a Model 10-AU fluo- kinetics controlling the observed rate (Ahlgren 1987). rometer (Turner Designs, Sunnyvale, CA), or by cell Under such conditions, biochemical processes are counts (see next subsection). All sampling was per- expected to have a Q10 near 2. There are relatively few formed on mid-log-phase cultures. Under these condi- examples where the effects of temperature on physio- tions, growth can be maintained at a constant rate vir- logical and biochemical processes have actually been tually indefinitely, and cell composition is essentially examined simultaneously. invariant over time (e.g. Berges & Harrison 1995a,b). Previous work considering the effects of temperature Cell composition measurements. Cell counts and on nitrogen metabolism is somewhat disjointed. We cell volume determinations were performed on living know, for example, that nitrogen uptake can show a cells using a Model TAII counter (Beckmann Coulter, pattern of increase, optimum and rapid decline as Brea, CA) equipped with a population accessory. A temperature is increased (Rhee & Gotham 1981, Terry 70 µm aperture was used, and the instrument was cal- 1983, Whalen & Alexander 1984), that there can be a ibrated with 5 µm latex microspheres. Chl a was mea- pronounced uncoupling between transient uptake and sured fluorometrically in samples collected by filtration assimilation of nitrogen at lower temperatures (Raim- onto 25 mm GF/F glass-fibre filters, and extracted in bault 1984), and that the activity of nitrogen-reducing 90% acetone (Parsons et al. 1984). Samples taken on enzymes such as nitrate reductase (NR) varies strongly precombusted 13 mm Gelman A/E filters were ana- with temperature (Kristiansen 1983). Recent work by lyzed for nitrogen and carbon content using a CNS Gao et al. (2000) provided some evidence of the bio- analyzer (Carlo Erba, Milan, Italy), with sulfanilamide chemical basis for the observations of Lomas & Glibert as a standard. Protein was determined using a modi- (1999a,b); NR from the diatom Skeletonema costatum fied Bradford method (described in Berges et al. 1993), shows optimal activity at a relatively low temperature homogenizing samples filtered onto 25 mm GF/F filters and is unstable above ~16°C. in TCA, and resolublizing proteins in 1 N NaOH. In the present study, we examined the effects of tem- Nitrogen metabolism. Nitrate uptake rates were perature on cell composition, nitrate uptake and incor- measured using the stable isotope 15N. Duplicate sam- poration, nitrate reduction (catalyzed by NR) and ulti- ples from each culture were inoculated with 10 µM 15 – mately on rates of growth, represented by the rate of NO3 and incubated at 8, 17 or 25°C alongside the cell division rate, in near-steady-state cultures of the original cultures in 500 ml polycarbonate bottles for 3 marine diatom Thalassiosira pseudonana. This repre- to 4 h. After incubation, samples for 15N analysis were sents one of the first attempts to compare ecological, collected on precombusted GF/F filters and analyzed physiological and biochemical rates to deduce at what using the micro-Dumas dry-combustion technique, as Berges et al.: Temperature effects on Thalassiosira pseudonana 141

described by La Roche (1983) and Harrison (1983), and ) ) -1 a Model N-150 (Jasco International, Tokyo, Japan) 16 A -1 3.6 B emission spectrometer (Fiedler & Proksch 1975). Nitrate uptake rates were calculated following Dug- 3.2 12 dale & Wilkerson (1986). 2.8 In vitro assays for nitrate reductase (NR) were per- on (pg cell

8 cell (pg Protein 2.4 formed as described in Berges & Harrison (1995a), Carb monitoring production of nitrite colourimetrically. Duplicate samples from each culture were collected 812162024 812162024 on GF/F filters and homogenized. Subsamples of each

homogenate were assayed at all temperature, i.e. 8, 17 ) -1 CD0.4 and 25°C. 2.0 ) -1 Rates of nitrate incorporation in cultures at the tem- cell 0.3 perature of acclimation were also calculated as the pg 1.6

product of specific growth rate (µ) and particulate (pgcell 0.2 a nitrogen per cell (PN). ogen ( 1.2 15 Chl Chl Analyses. For rates of growth, N uptake, NR activ- Nitr 0.1 ity and calculated nitrate incorporation, values of Q10 and activation energy (Ea) were calculated across tem- 812162024 812162024 perature intervals as:

− ) 10(lnVV21 ln ) 3 lnQ10 = 10 EF52 ()TT21− m µ and 48

RV(ln− ln V ) (mol:mol) 8 lnE = 21 a − 44 ()TT12 where V1 and V2 are rates of reaction at temperatures 6 40 Cell volume ( Cell volume T1 and T2 (in °K), and R is the gas constant. C:N ratio Comparisons of growth rates, cell composition and rates of nitrogen metabolism at different temperatures 812162024 812162024 were made using 1-way ANOVA designs followed by Tukey multiple comparisons, using SigmaStat Version Fig. 1. Thalassiosira pseudonana. Cell constituents in cultures 1.0 (Jandel Scientific, San Rafael, CA); all tests were grown under continuous saturating irradiance at different made at the 95% confidence level. temperatures. (A) particulate carbon; (B) protein; (C) particulate nitrogen; (D) chl a; (E) carbon:nitrogen molar ratio; (F) cell volume. Cells were grown in semi-continuous batch cultures and acclimated to each temperature for a minimum RESULTS of 8 generations before sampling; all cultures were in mid- logarithmic phase when sampled. Each point represents the In terms of cell composition, there were no signifi- mean (±SE) of 3 replicate cultures; where error bars are not visible, they are smaller than the symbol cant differences in particulate nitrogen (Fig. 1C; p > 0.1), protein (Fig. 1B; p > 0.2) or cell volume (Fig. 1F; p> 0.4) among different temperatures. Particulate car- Table 1. Thalassiosira pseudonana. Values of Q10 and appar- bon per cell increased with increasing temperature –1 ent activation energy (Ea, kJ mol , in parentheses) for growth (Fig. 1A; p < 0.05), as did the C:N ratio (Fig. 1E; p < rate (µ), 15N uptake, and NR activity and calculated nitrate 0.01) and chl a per cell (Fig. 1D; p < 0.01); the magni- incorporation rate (the product of µ and particulate nitrogen tude of increases between 17 and 25°C was greater per cell, PN) over different temperature ranges, for diatom cultures. Variables were measured in triplicate log-phase than those between 8 and 17°C. cultures acclimated to the temperature of measurement for For rate measurements, Q10 values differed substan- a minimum of 8 generations. Q10 and Ea were calculated as tially depending on the temperature interval used for described in last subsection of ‘Materials and methods’ the calculation. In general, rates calculated over the whole range from 8 to 25°C tended to be near 2, while Variables 8 to 17°C 17 to 25°C 8 to 25°C those from 8 to 17°C were greater than 2 (Table 1). Be- tween 17 and 25°C, some rates (15N uptake and NR ac- µ 3.09 (76.3) 1.19 (12.6) 1.97 (46.0) 15N uptake 2.88 (71.6) 0.98 (–0.95) 1.65 (33.9) tivity) actually declined, resulting in Q values of <1.0. 10 NR activity 2.71 (67.3) 0.95 (–3.9) 1.74 (37.5) Growth rate increased between 8 and 17°C with a Q10 µPN 3.18 (78.2) 1.63 (35.1) 2.32 (57.0) of >3, but there was a substantially smaller change be- 142 Mar Ecol Prog Ser 225: 139–146, 2002

17°C (Table 1). Between 17 and 25°C, Q10 and Ea for ) 15 -1 2.0 A NR activity and N uptake rates were no different (d

µ from each other (p > 0.1); although rate constants for 1.5 µPN appeared to be higher than for 15N uptake rates or

oration NR activities (Table 1), these differences were not 1.0 ) statistically significant (p > 0.05). 12

N incorp When NR activity was plotted against assay temper- 0.5 x 10 15 ) ature, trends were similar for cultures grown at differ- -1

cell ent temperatures (Fig. 3). A 2-way ANOVA examining

-1 160 the effects of growth temperature and assay tempera- PN or ( or PN B µ ) ture showed no significant differences between NR 120 activity of cultures that were grown at different tem- mol N min µ ( peratures and assayed at a common temperature (p > 80 0.1), and no interactions between growth temperature NR activity, ( NR activity, ) and assay temperature (p > 0.1). Significant differ- ( 40 ences were found between assay temperatures (p < 0.05), regardless of the temperature at which cultures 812162024 had been grown; NR activity was lower at 8°C and not Temperature (°C) significantly different between 17 and 25°C. Fig. 2. Thalassiosira pseudonana. Rates of growth (A) and indices of nitrogen incorporation (B) in cultures grown under continuous saturating irradiance at different temperatures. Growth DISCUSSION was calculated from changes in cell number. Nitrogen incorporation was estimated as nitrate reductase (NR) activity (Z), rate of nitrogen incorporation as the product of specific growth rate Temperature and cell composition (µ), and cell nitrogen content (PN, j), or rate of 15N-nitrate uptake (M). Culture conditions and sampling as in Fig. 1 The results for cell composition are largely consistent with previous findings: increasing C content and chl a content with increasing temperature have been noted tween 17 and 25°C (Fig. 2A, Table 1). NR activity, 15N many times before for algae in general (see Thompson uptake and calculated rates of nitrate incorporation et al. 1992), and diatoms in particular (e.g. Lomas & (µPN) were not significantly different from each other Glibert 1999a, Gao et al. 2000). There is more variabil- at any temperature (Fig. 2B; p > 0.05 in all cases). In ity in the results for cell volume. In some cases, increas- terms of rate constants, the Q10 and Ea for NR activity, ing volume with increasing temperature has been 15N uptake and calculated rates of nitrate incorporation noted by some investigators (e.g. Lomas & Glibert were very close to those for growth rate between 8 and 1999a), while others have found no changes (e.g. Gao et al. 2000). This may be related to the light regime: cell volume increases tend to occur in studies using a light:dark cycle (e.g. Lomas & Glibert 1999a), as

) 8 °C opposed to continuous light (as in the present study). 12 17 °C 120 The finding that C:N ratio increases with increasing

x 10 °

25 C

-1 temperature is generally supported by other data (e.g. cell

Thompson et al. 1992), but this may also depend on the -1 light regime, since Lomas & Glibert (1999a) noted no 80 changes in C:N ratio with temperature in diatoms and

mol Nmol min dinoflagellates grown on light:dark cycles. In the pre- µ sent study, the increase in C:N ratio was largely the ty ( 40 result of increased carbon content without changes in nitrogen. Yoder (1979) found that in the diatom Skele- NR activi tonema costatum, N content was largely independent

812162024 of temperature under light limitation, and this seems also to be the case where light is not limiting. The Assay temperature (°C) mechanism of this change remains unclear. Raven & Fig. 3. Thalassiosira pseudonana. Nitrate reductase (NR) activity in cultures grown at 8, 17 or 25°C and assayed at differ- Geider (1988) hypothesized that algae grown at low ent temperatures. Culture conditions, sampling and symbols temperature could commit a larger fraction of cell car- as in Fig. 1 bon to catalysts than algae at higher temperatures. If Berges et al.: Temperature effects on Thalassiosira pseudonana 143

so, assuming that catalysts are enzyme proteins, we perature in which plants were capable of growing). might expect that low-temperature-acclimated cells Such a concept has not been applied to phytoplankton would have relatively more carbon tied up in protein to our knowledge. than those at higher temperatures, and thus the C:N ratio would be lower at low temperatures. However, were this the case, we might also expect to see chan- Temperature effects on growth ges in protein, which were not observed. Similarly, Gao et al. (2000) did not detect any changes in cell pro- Considerable data are available on the effects of tein in S. costatum grown at temperatures between 5 temperature on algal growth; however, results are usu- and 25°C. Alternatively, temperature could affect rates ally based either on changes in the rate of cell division of carbon and nitrogen incorporation differentially. (i.e. µ, largely in laboratory studies), or on rates of carbon fixation (largely in fieldwork). In order to compare our results across these data sets, we make the

The Q10 concept assumption that growth and photosynthesis are closely linked and that cell composition is relatively constant. Before comparing the results of the various measure- While such assumptions are almost certainly false in ments of rates of growth and nitrogen metabolism, it is particular cases (see preceeding subsection), it none- necessary to evaluate the Q10 and Ea calculations. Both theless appears that both growth and photosynthetic measurements varied considerably, depending on the rates respond to temperature with an apparent Q10 temperature interval chosen. Correct use of a Q10 value near 2 (Eppley 1972, Raven & Geider 1988, Davison implies that the data follows an Arrhenius-type rela- 1991); a more precise value of 1.88 is often quoted (see tionship (linearity of a log vs inverse absolute tempera- Raven & Geider 1988). If the whole temperature range ture). Several authors have argued against such func- of growth rate data is considered in the present study, tions, advocating Belehradek or empirical square-root a quite similar value of Q10 (1.97) is found, but this dif- relationships instead (see Ratkowsy et al. 1983, Ahl- fers considerably depending on the temperature inter- gren 1987). One of the important failings of the Q10 val chosen. In general, diatoms do not appear to be concept is that it is almost certain that different pro- exceptional to the general rule for algae. Lomas & cesses become limiting at different temperature (see Glibert (1999a) found a Q10 of 2.46 for Thalassiosira Jumars et al. 1993). For example, for photosynthesis, weissflogii grown between 10 and 20°C, and Smith et photosynthetic electron transport is probably limiting al. (1994) and Suzuki & Takahashi (1995) quote values at lower temperatures, while processes related to in the neighbourhood of 1.9 for a variety of other spe- transport and fixation of carbon are probably limiting cies. As in the present study, Thompson et al. (1992) at higher temperatures (see Davison 1991). noted for T. pseudonana that values of Q10 varied However, rather than simply abandoning the use of between 1.8 and 3.1, depending on the particular

Q10 as a means to describe and predict temperature range of temperatures selected. Interestingly, Suzuki & effects on metabolic rates, we suggest that Q10 could Takahashi noted, for 8 Arctic diatom species, that the be used in a more fundamental way to provide infor- temperatures at which maximal growth rates were mation about the temperatures to which organisms are found were all very near the upper limit for growth and adapted. Such an idea is, in fact, quite common in the generally higher than the temperature from which the zoological literature; for example, Brown (1989) de- species were isolated. This suggests that temperature fined the ranges of temperature in which fishes ex- could be an important factor for these species and that perienced thermal stress as the ranges of temperature the range of growth itself is less important ecologically in which oxygen consumption changed by a Q10 >4. than some smaller subset of this range (see preceeding Such a region could be defined for algae based on subsection). growth rate: Jitts et al. (1964) examined cell division of It has been noted for photosynthesis that the stability 5 species of microalgae and was able to identify ranges of enzymes associated with carbon fixation (e.g. ribu- of 6 to 10°C, outside of which the Q10 for growth varied lose bisphosphate carboxylase/oxygenase: RuBisCO) considerably around 2. Such a region could be defined generally exceeds that of whole-plant photosynthesis, for enzyme activities as well; Burke (1995) identified i.e. enzymes are less temperature-sensitive than the regions of temperature stability based on enzyme integrated process in which they participate (see Davi- kinetics that he termed a ‘thermal kinetic window’ son 1991). Moreover, Devos et al. (1998) showed that,

(TKW), i.e. the range in which the effective Km for an in a number of Chloromonas species (both psychro- enzyme was within 200% of the minimum observed. philes and species adapted to higher temperatures), For higher plants, this was usually in the 5 to 8°C growth rates, rates of photosynthesis and activities ranges (and was thus much less than the range of tem- of Rubisco have similar thermal optima and Rubisco 144 Mar Ecol Prog Ser 225: 139–146, 2002

enzymes from different isolates have similar thermo- difference has been discussed in an evolutionary con- stability. Such findings contrast with the case for NR text (Gao et al. 2000). Intriguingly, Kudo et al. (2000) (see next subsection). have recently demonstrated that NR from Phaeodacty- lum tricornutum shows very little variation in activity with changing temperature. This result contrasts with Temperature effects on nitrate metabolism many other studies in diatoms (e.g. Kristiansen 1983 and Gao et al. 2000), and may indicate fundamental Effects of temperature on NR activities and other differences between P. tricornutum and other species. indices of nitrate incorporation appear to be consistent with each other, within the errors of the measurements. While such correlations cannot prove causa- Thermal regulation of NR activity tion, the results do support the hypothesis that temperature effects on nitrate metabolism are mediated How is the effect of temperature on diatom NR medi- at the enzyme level. Of particular interest is the lack ated? Based on previous work, NR activity in diatoms of change in nitrate metabolism between 17 and 25°C; such as Thalassiosira weissflogii appears to be regu- directly measured rates of 15N uptake and NR activity lated largely by synthesis (e.g. Gao et al. 2000); to a are virtually unchanged, and although rates calcu- first approximation, a continuous background degra- lated from N content and cell division rates do appear dation accomplishes its turnover (see Vergara et al. to increase, they are not statistically different. This is 1998). In contrast to NR, which is rather labile in evidence that the processes of N acquisition are more diatoms (e.g. Kristiansen 1983, Gao et al. 1993), prote- sensitive to temperature than are cell division and olytic enzymes in phytoplankton are remarkably ther- photosynthesis in diatoms, and fits very nicely with mostable (Berges & Falkowski 1996). We can hypothe- Lomas & Glibert’s (1999a,b) hypothesis. More re- size that, as temperature increases, the activities of the cently, Lomas & Glibert (2000) have provided addi- proteases that normally degrade NR also increase. tional evidence that the critical step is that catalyzed Thus, without any more complicated cellular regula- by NR and not nitrite reductase (NiR). Their work has tion, increased temperature should lead to greater also highlighted differences between diatoms and degradation of NR and not necessarily to greater syn- flagellate species that can be related to differing tem- thesis; lower NR activity could simply be a conse- perature optima for NR. quence of these features of the enzyme’s regulation. In reviewing the algal literature, it appears that tem- In reality, regulation of NR may be much more com- perature optima and general characteristics of nitrate plicated. Simply changing the quantity (and thus activ- uptake and NR activities tend to be very similar in a ity) of enzymes is a relatively poor strategy for dealing variety of species including cyanobacteria (e.g. Shukla with temperature changes; altering enzyme kinetic

& Kashyap 1999) and dinoflagellates (Kristiansen 1983, constants (e.g. Km) through post-translational modifi- Witt et al. 1999). Temperature responses of NR are also cation, or through temperature-specific expression of similar: Witt et al. (1999) found Ea values for NR from isozymes are more efficient and often-used strategies Peridinium gatunense of 23 to 29 kJ mol–1; Kristiansen (see Graham & Patterson 1982, Burke 1995). With –1 (1983), derived an Ea of 38 kJ mol for NR from Hete- respect to algal nitrogen metabolism, nothing is yet rocapsa triqueta; and Gao et al. (1993) calculated an Ea known about such strategies. of 36.5 kJ mol–1 for NR from Skeletonema costatum, all As mentioned above, Gao et al. (2000) hypothesised very similar to the overall value found in the present that there are significant evolutionary differences be- study. tween NR from green algae and higher plants and NR NR activity from a wide number of marine species from chromophytes. One way in which this manifests it- from lineages containing chl c show thermal optima in self is in the light regulation of NR: higher plants show a the range of 10 to 20°C (e.g. Kristiansen 1983, Davison phosphorylation mechanism (Huber et al. 1992), while & Davison 1987, Gao et al. 1993), Interestingly, all the diatoms do not (Berges 1997, Vergara et al. 1998). Curi- available data for diatom NR seem to suggest very sim- ously, earlier work (Nussaume et al. 1995) demon- ilar thermal optima for the enzyme, near 16°C. This strated that NR from higher plants carries an N-termi- temperature is not well related to the temperature from nal domain that is not conserved in other organisms. which the phytoplankton have been isolated (see When the N-terminal domain is removed from tobacco Packard et al. 1971, Kristiansen 1983, Gao et al. 1993), NR, not only is post-translational light regulation abol- and this remains something of a puzzle. Such an opti- ished, but the thermal optimum of the enzyme shifts mum is much lower than that found for green algae from 30 down to near 15°C. We have no diatom NR se- and higher plants (e.g. Solomonson & Vennesland 1972, quences at present but, when they are available, it will Solomonson & Barber 1990, Loppes et al. 1996). This be interesting to compare N-terminal sequences. Berges et al.: Temperature effects on Thalassiosira pseudonana 145

Acknowledgements. We thank Steve Ruskie for valuable nitrate reductase activity in marine phytoplankton: bio- technical assistance. Comment from Mike Lomas and 2 chemical analysis and ecological implications. J Phycol 36: anonymous referees substantially improved the manuscript. 304–313 J.A.B. was supported in part by a Killham pre-doctoral Gibson CE, Foy RH (1989) On temperature independent fellowship. growth of phytoplankton. J Plankton Res 11:605–607 Graham D, Patterson DB (1982) Responses of plants to low, non-freezing temperatures: proteins, metabolism and LITERATURE CITED acclimation. Annu Rev Plant Physiol 33:347–372 Harrison WG (1983) Nitrogen in the marine environment: use Ahlgren G (1987) Temperature functions in biology and of isotopes. In: Capone D, Carpenter E (eds) Nitrogen in their application to algal growth constants. Oikos 49: the marine environment. Academic Press, New York, 177–190 p 763–807 Behrenfeld M, Falkowski P (1997) A consumer’s guide to Huber JL, Huber SC, Campbell WH, Redinbaugh MG (1992) phytoplankton primary productivity models. Limnol Reversible light/dark modulation of spinach leaf nitrate Oceanogr 42:1479–1491 reductase activity involves protein phosphorylation. Arch Berges JA (1997) Algal nitrate reductases. Eur J Phycol 32: Biochem Biophys 296:58–65 3–8 Jitts H, McAllister C, Stephens K, Strickland J (1964) The cell Berges JA, Falkowski PG (1996) Cell-associated proteolytic division rates of some marine phytoplankters as a function enzymes from marine phytoplankton. J Phycol 32:566–574 of light and temperature. J Fish Res Board Can 21:139–157 Berges JA, Harrison PJ (1993) Relationship between nucleo- Jumars P, Deming J, Hill P, Karp-Boss L, Yager P, Dade W side diphosphate kinase activity and light-limited growth (1993) Physical constraints on marine osmotrophy in an rate in the marine diatom Thalassiosira pseudonana optimal foraging context. Mar Microb Food Webs 7: (Bacillariophyceae). J Phycol 29:45–53 121–159 Berges JA, Harrison PJ (1995a) Nitrate reductase activity Kristiansen S (1983) The temperature optimum of the nitrate quantitatively predicts the rate of nitrate incorporation redutase assay for marine phytoplankton. Limnol Ocea- under steady state light limitation: a revised assay and nogr 28:776–780 characterization of the enzyme in three species of marine Kudo I, Miyamoto M, Noiri Y, Maita Y (2000) Combined phytoplankton. Limnol Oceanogr 40:82–93 effects of temperature and iron on the growth and physio- Berges JA, Harrison PJ (1995b) Relationships between nitrate logy of the marine diatom Phaeodactylum tricornutum reductase activity and rates of growth and nitrate incorpo- (Bacillariophyceae). J Phycol 36:1096–1102 ration under steady-state light or nitrate limitation in the La Roche J (1983) Ammonium regeneration: its contribution marine diatom Thalassiosira pseudonana (Bacillariophy- to phytoplankton nitrogen requirements in a eutrophic ceae). J Phycol 31:85–95 environment. Mar Biol 75:231–240 Berges JA, Fisher AE, Harrison PJ (1993) A comparison of Li WKW (1980) Temperature adaptation in phytoplankton: Lowry, Bradford, and Smith protein assays using different cellular and photosynthetic characteristics. In: Falkowski protein standards and protein purified from the marine PG (ed) Primary productivity in the sea. Plenum Press, diatom Thalassiosira pseudonana. Mar Biol 115:187–193 New York, p 259–279 + Brown LR (1989) Temperature preferences and oxygen con- Lomas MW, Glibert PM (1999a) Interactions between NH4 – sumption of three species of Sculpin (cottus) from the Pit and NO3 uptake and assimilation: comparisons of diatoms River drainage California USA. Environ Biol Fish 26: and dinoflagellates at several growth temperatures. Mar 223–226 Biol 133:541–551 Burke J (1995) Enzyme adaptation to temperature. In: Lomas MW, Glibert PM (1999b) Temperature regulation of Smirnoff N (ed) Environment and plant metabolism: nitrate uptake: a novel hypothesis about nitrate uptake flexibility and acclimation. Bios Scientific Publishers, and reduction in cool-water diatoms. Limnol Oceanogr 44: Oxford, p 63–79 556–572 Davison IR (1991) Environmental effects on algal photosyn- Lomas MW, Glibert PM (2000) Comparisons of nitrate uptake, thesis: temperature. J Phycol 27:2–8 storage, and reduction in marine diatoms and flagellates. Davison IR, Davison JO (1987) The effect of growth tempera- J Phycol 36:903–913 ture on enzyme activities in the brown alga Laminaria sac- Loppes R, Devos N, Willem S, Barthelemy P, Matagne RF charina. Br J Phycol 22:77–87 (1996) Effect of temperature on two enzymes from a Devos N, Ingouff M, Loppes R, Matagne RF (1998) Rubsico psychrophilic Chloromonas (Chlorophyta). J Phycol 32: adaptation to low temperatures: a comparative study in 276–278 psychrophili and mesophilic unicellular algae. J Phycol 34: Morris I, Glover HE, Yentsch CS (1974) Products of photo- 655–660 synthesis by marine phytoplankton: the effect of environ- Dugdale RC, Wilkerson FP (1986) The use of 15N to measure mental factors on the relative rates of protein synthesis. nitrogen uptake in eutrophic oceans; experimental consid- Mar Biol 27:1–9 erations. Limnol Oceanogr 31:673–689 Nussaume L, Vincentz M, Meyer C, Boutin JP, Caboche M Eppley R (1972) Temperature and phytoplankton growth in (1995) Post-translational regulation of nitrate reductase by the sea. Fish Bull 70:1063–1085 light is abolished by an N-terminal deletion. Plant Cell Fiedler R, Proksch G (1975) The determination of nitrogen-15 7:611–621 by emission and mass spectrometry in biochemical analy- Packard TT, Blasco D, MacIsaac JJ, Dugdale RC (1971) Vari- sis: a review. Anal Chim Acta 78:1–62 ations of nitrate reductase activity in marine phytoplank- Gao Y, Smith GJ, Alberte RS (1993) Nitrate reductase from ton. Invest Pesq 35:209–219 the marine diatom Skeletonema costatum. Biochem- Parsons T, Maita M, Lalli C (1984) A manual of chemical and ical and immunological characterization. Plant Physiol biological methods for seawater analysis. Pergamon Press, (Bethesda) 103:1437–1445 Oxford Gao Y, Smith G, Alberte R (2000) Temperature dependence of Raimbault P (1984) Influence of temperature on the transient 146 Mar Ecol Prog Ser 225: 139–146, 2002

response in nitrate uptake and reduction by four marine Solomonson LP, Barber MJ (1990) Assimilatory nitrate reduc- diatoms. J Exp Mar Biol Ecol 84:37–53 tase: functional properties and regulation. Annu Rev Plant Ratkowsky D, Lowry R, McMeekin T, Stokes A, Chandler R Physiol Plant Mol Biol 41:225–253 (1983) Model for bacterial culture growth rate throughout Solomonson LP, Vennesland B (1972) Properties of a nitrate the entire biokinetic temperature range. J Bacteriol 154: reductase of Chlorella. Biochim Biophys Acta 267:544–557 1222–1226 Suzuki Y, Takahashi M (1995) Growth responses of several Raven JA, Geider RJ (1988) Temperature and algal growth. diatom species isolated from various environments to tem- New Phytol 110:441–461 perature. J Phycol 31:880–888 Reay DS, Nedwell DB, Priddle J, Ellis-Evans JC (1999) Tem- Terry K (1983) Temperature dependence of ammonium and perature dependence of inorganic nitrogen uptake: reduced phosphate uptake and their interaction in the marine affinity for nitrate at suboptimal temperatures in both algae diatom Phaeodactylum tricornutm Bohlin. Mar Biol Lett 4: and bacteria. Appl Environ Microbiol 65:2577–2584 309–320 Rhee GY, Gotham IJ (1981) The effect of environmental fac- Thompson PA, Guo M, Harrison PJ (1992) Effects of variation tors on phytoplankton growth: temperature and the inter- in temperature. I. On the biochemical composition of eight actions of temperature with nutrient limitation. Limnol species of marine phytoplankton. J Phycol 28:481–488 Oceanogr 26:635–648 Vergara JJ, Berges JA, Falkowski PG (1998) Diel periodicity Sakamoto T, Bryant DA (1999) Nitrate transport and not pho- of nitrate reductase activity and protein levels in the ma- toinhibition limits growth of the freshwater cyanobac- rine diatom Thalassiosira weissflogii (Bacillariophyceae). terium Synechococcus species PCC 6301 at low tempera- J Phycol 34:952–961 ture. Plant Physiol (Bethesda) 119:785–794 Whalen SC, Alexander V (1984) Influence of temperature and Shukla SP, Kashyap AK (1999) The thermal responses and light on rates of inorganic nitrogen transport by algae in activation energy of PSII, nitrate uptake and nitrate reduc- an Arctic lake. Can J Fish Aquat Sci 41:1310–1318 tase activities of two geographically different isolates of Witt FG, Stohr C, Ullrich WR (1999) Soluble and membrane- Anabena. Cytobios 99:7–17 associated nitrate reductases in the dinoflagellate Peri- Smith R, Stapleford L, Ridings R (1994) The acclimated dinium gatunense. New Phytol 142:27–34 response of growth, photosynthesis, composition, and car- Yoder J (1979) Effect of temperature on light-limited growth bon balance to temperature in the psychrophilic ice and chemical composition of Skeletonema costatum (Ba- diatom Nitzschia seriata. J Phycol 30:8–16 cillariophyceae). J Phycol 15:362–370

Editorial responsibility: Otto Kinne (Editor), Submitted: October 16, 2000; Accepted: April 19, 2001 Oldendorf/Luhe, Germany Proofs received from author(s): January 8, 2002